Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland. smaragda.lamprianou@unige.ch

Abstract

OBJECTIVE:

We studied whether manganese-enhanced high-field magneticresonance (MR) imaging (MEHFMRI) could quantitatively detect individualislets in situ and in vivo and evaluate changes in a model of experimental diabetes.

RESEARCH DESIGN AND METHODS:

Whole pancreata from untreated (n = 3), MnCl(2) and glucose-injected mice (n = 6), and mice injected with either streptozotocin (STZ; n = 4) or citrate buffer (n = 4) were imaged ex vivo for unambiguous evaluation of islets. Exteriorized pancreata of MnCl(2) and glucose-injected mice (n = 6) were imaged in vivo to directly visualize the gland and minimize movements. In all cases, MR images were acquired in a 14.1 Tesla scanner and correlated with the corresponding (immuno)histological sections.

RESULTS:

In ex vivo experiments, MEHFMRI distinguished different pancreatic tissues and evaluated the relative abundance of islets in the pancreata of normoglycemic mice. MEHFMRI also detected a significant decrease in the numerical and volume density of islets in STZ-injected mice. However, in the latter measurements the loss of β-cells was undervalued under the conditions tested. The experiments on the externalized pancreata confirmed that MEHFMRI could visualize native individualislets in living, anesthetized mice.

CONCLUSIONS:

Data show that MEHFMRI quantitatively visualizes individualislets in the intact mouse pancreas, both ex vivo and in vivo.

High-resolution MRI reveals the structure of the entire mouse pancreas. A and B: Whole mouse pancreata (300-μm-thick slices) were imaged ex vivo, with 50 μm in plane resolution (TR/TE = 282/7 ms). A: In an image obtained without contrast agent, pancreatic lobules are seen, most of which feature a somewhat homogeneous and structureless content at this low magnification. Scale bar: 1 mm. B: Under these conditions, MRI infrequently identified putative islets of Langerhans (green arrows) within the intact pancreas. Scale bar: 1 mm. C: The contrast of pancreatic lobules was enhanced after intravenous infusion of MnCl2 combined with an intraperitoneal injection of glucose. Under these conditions, MRI allowed to distinguish whitish tubular structures, as well as highly contrasted round-ovoid structures of various sizes. The smallest of these structures (<0.5 mm; green arrows) was observed within the pancreatic lobules. Larger structures (>1 mm) were seen between the lobules. The latter structures were identified by histological analysis of the very same pancreas, confirming that MRI differentiates the pancreatic parenchyma from intrapancreatic lymphatic ganglia (C1), spleen (C2), and loops of small intestine (C3). Scale bar: 1 mm in A–C; 0.5 mm in lower panels B1–3. (A high-quality digital representation of this figure is available in the online issue.)

Individualpancreaticislets are visualized by MRI. A: This MR image (left) is a high magnification view of the pancreas shown in , recorded with a 50 μm in plane resolution. Small, round-ovoid whitish structures (several are pointed by green arrows) are seen dispersed within the pancreatic lobules. Histology (right) confirmed that these structures are pancreaticislets of Langerhans, dispersed within the exocrine parenchyma, between vessels and ducts (white arrows). Scale bar: 1 mm. B: Quantification of the islet signal intensity, relative to that of the surrounding exocrine tissue, confirmed that the MR image of islets was more contrasted (***P < 0.001) in the Mn2+-infused mice (red bar) than in noninfused controls (blue bar). Values are means + SEM of the number of islets indicated within the columns. a.u., Arbitrary units. (A high-quality digital representation of this figure is available in the online issue.)

MRI quantifies islet mass and size. A: Islets of different sizes were detected in the MR images of whole mouse pancreas (top) and confirmed by correlative histology (bottom). The right panels are enlargements of the regions boxed in the left panels. Scale bar: 1 mm. B: Islet sizes were measured in both MR and histology images. The distribution of the values was asymmetrical in both cases, confirming that islets <150 μm in diameter predominate in the control mouse pancreas, whereas islets >300 μm are rare. Of note, islets <50 μm in diameter escaped MRI detection. C: Further morphometric evaluation of MR images showed that MRI detected on the average 1 islet/mm3 of pancreas (left), accounting for a relative volume of the endocrine micro organs of 0.8 ± 0.2% (middle). A mean of 268 islets per animal (n = 5) was detected; Vvi, volume density of islets; Nvi, volume density of the islets (Ni/mm3 of pancreas); Vp, total volume of pancreas analyzed (mm3). Values are means + SEM of five control mice. (A high-quality digital representation of this figure is available in the online issue.)

MRI detects the loss of islets in a diabetic mouse model. A: High magnification MR and histology images of pancreas from mice injected with either CB (normoglycemic controls) or the buffer supplemented with STZ (hyperglycemic mice). Whitish (MRI)/pink islets (histology) were seen more frequently in the pancreata of control rather than of STZ-injected mice (some islets are pointed by green arrows). Scale bar: 1 mm and 0.5 mm in the insets. B: Morphometric analysis of the MR images showed that hyperglycemic mice had a pancreas volume (Vp; bottom) that was not significantly different from that of the citrate-injected controls. In contrast, the relative number (Nvi; top) and volume density (Vvi; middle) of the islets were decreased by 55 and 45%, respectively, in the STZ-injected mice. Data are mean + SEM of the indicated number of pancreas (**P < 0.01; *** P < 0.001). C: Immunostainings of consecutive pancreas sections of control and STZ-treated mice with antibodies against insulin and glucagon show that the STZ treatment significantly reduced the number of β-cells in most islets (outlined by white dotted lines). Most islets still contained many glucagon-containing α-cells. Some small islets (arrows) were essentially made by the latter type of islet cells and were fully devoid of insulin-containing cells. Scale bar: 0.5 mm. (A high-quality digital representation of this figure is available in the online issue.)

MRI detectsislets in situ in a living mouse. High magnifications of MR images of the exteriorized pancreas of a living, anesthetized mouse reveal a pancreatic substructure like that observed ex vivo, including the presence of elongated vessels and ducts (white arrows) and round-ovoid bodies of small size (green arrows). Histological correlation showed that these small bodies corresponded to individualpancreaticislets. Scale bar: 1 mm. Insets show low magnification views of the same pancreas. P = pancreas; s = spleen. *Position of the plastic pin that secured the pancreas. (A high-quality digital representation of this figure is available in the online issue.)